Thymidylate Synthase (TS) catalyzes the conversion of dUMP and 5,10-CH2-H4folate into dTMP. It is the only known path for dTMP synthesis. dTMP synthesis is believed to be one of the rate limiting steps in DNA replication. The inhibitor of this enzyme can be used to block dTMP synthesis and can be of possible chemotherapeutic use for cancers. We performed Free Energy Perturbation (FEP) calculations on selected molecules with different combinations of substitutions of inhibitors. The calculation results agree with experimental values for selected inhibitors that lack glutamate residues. Most of these compounds have the same N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline structure. The aniline was substituted with simple lipophilic substituents at different positions. A set of Pictorial Representation of Free Energy Change (PROFEC) calculations was also performed. We found that fluorine-substitution at the R5 and R6 positions enhances binding. The most encouraging result is that one compound shows about 6 Kcal/mole binding enhancement. It suggested that R5 or R6 substitution could be a new direction for the design of TS inhibitors. The Computer Graphics Laboratory is crucial in designing the inhibitors and viewing the PROFEC results, especially the 3D ability.